Polyubiquitin (polyUb) chains linked through Ub-K63 act as a non-proteolytic signal in the conserved RAD6 DNA damage tolerance pathway, while chains linked through Ub-K48 play a different role, acting as a signal for proteasome proteolysis. These and other observations suggest that the assembly of Ub into different types of polyUb chains is a mechanism for imparting functional diversity in signaling by ubiquitin. Signaling by K63-1inked polyUb chains in DNA damage tolerance is conserved from yeast to man. But despite recent advances, the molecular function of this novel signal remains poorly understood. The proposed research targets this question through mechanistically focused approaches involving biochemical and molecular genetic methods. The specific goals are 1) to elucidate the specific mechanism of DNA damage signaling by K63-1inked polyUb chains (Aims 1-3) and 2) to discover the principles that underlie linkage-specific chain recognition by a small Ub-binding element that occurs in numerous proteins (the UBA domain; Aim 4). The results of our studies may shed light on the causes of cancer and other diseases promoted by genomic instability. Our findings may also suggest ways to inhibit the RAD6 pathway, which could be clinically beneficial in chemotherapy or radiosensitization. Finally, our molecular exploration of (poly)Ub recognition by UBA domains should help to establish fundamental signal recognition principles and may lead to a better appreciation of diversity and specificity in signaling by ubiquitin.